DNA amplification is frequently used in DNA diagnostic tests and sequencing protocols. Both require a low background of non-specific amplification. Unfortunately, amplification methods that utilize primers appear to give rise to false positive signals of non-template DNA that affect the quality of the results (Schlötterer and Tautz, Nucleic Acids Research, 20 (2):211-215 (1992); Ogata and Miura, Nucleic Acids Research, 26(20):4657-4661 (1998); Brukner, et al., Analytical Biochemistry, 339:345-347 (2005)). These false positive signals can be detected in control samples with non-template DNA or no DNA. Without wishing to be limited by theory, it is believed that false positives predominantly result from transient primer secondary structure formation and primer dimers.
Improvements in amplification signal and amplification specificity have not removed the adverse effects of background signal for isothermal amplification reactions. Chakrabati, et al., Nucleic Acids Research, 29:2377-2381 (2001), described enhancement of polymerase chain reaction (PCR) amplification using single low molecular weight amide additives. However, use of organic compounds is not compatible with all platforms.
Proteins and enzymes have been added in addition to the DNA polymerase for amplification reactions. These include single-stranded DNA binding proteins, clamps, and recombination proteins (Perales, et al, Nucleic Acids Research, 31(22):6473-6480 (2003); Fukui et al, Int J Mol Sci, 14:6436-6453 (2013)). Similarly, “hot start” approaches inhibit DNA polymerases in a temperature-dependent manner (U.S. Pat. No. 5,338,671). These methods may improve reaction performance but cannot eliminate non-template amplification at reaction temperatures.